We are interested in the transcriptional control of T cell development and function. Specifically, we study the gene expression programs that control the choice by intrathymic T cell precursors of the CD4 or CD8 lineage, and determine their distinctive functional responses. T cells are essential for immune responses. 'Conventional' T cells recognize peptide antigens presented by class I (MHC-I) or class II (MHC-II) classical Major Histocompatibility Complex molecules, and express either of two surface glycoproteins (called coreceptors) that contribute to antigen recognition: CD4, which binds MHC-II, or CD8, which binds MHC-I. Coreceptor expression on mature T cells is mutually exclusive and is essentially dictated by MHC specificity. That is, the general rule is that MHC I-specific T cells are CD4-CD8+ (CD8 cells), whereas MHC II-specific T cells are CD4+CD8- (CD4 cells). In addition, CD4 and CD8 T cells typically perform distinct functions upon antigen encounter: whereas CD8 T cells differentiate into cytotoxic effectors, CD4 T cells provide help to other components of the immune system (and of mucosal barriers) and have essential regulatory functions. Both the divergence of CD4 and CD8 lineages, and their pre-programming for helper and cytotoxic functions, respectively, occur in the thymus. We are interested in the development of CD4 T cells, which, because of their central role in immune responses, are essential to control infections (they are the key target of the human immunodeficiency virus HIV). Work conducted in the laboratory over the past 18 months has addressed two main questions: how do CD4 cells develop in the thymus and how is the integrity of the CD4 lineage maintained in mature T cells. Our previous studies had shown that the transcription factor Thpok is required for CD4 T cell differentiation in the thymus and notably represses CD8-lineage gene expression and CD8 T cell differentiation. More recently, we had shown that the combined activities of Thpok and the related transcription factor LRF (Leukemia-Lymphoma Related Factor) are essential for the emergence of helper effector functions in MHC II-restricted thymocytes. In a separate line of experiments, we had found that another transcription factor, Gata3, contributes both to promote Thpok expression and to restrain expression of CD8-lineage genes notably by repressing the gene encoding the transcription factor Runx3, a critical factor in CD8-lineage differentiation. We are currently pursuing investigations of the transcriptional circuitry driving CD4-lineage differentiation in the thymus by interrogating the functional relationships between Thpok, Gata3 and other factors involved in this process, including Tox and E-box binding proteins E2A and HEB. In parallel with these studies, we have addressed whether Thpok and LRF are important for the function of post-thymic (mature) of CD4 T cells. Both factors are expressed in essentially all mature CD4 T cells, as determined by flow cytometric analyses of intra-cellular protein expression or in 'reporter' mouse strains. In addition, the expression of Thpok in mature T cells remains specific of the CD4 lineage (whereas LRF is also expressed in CD8 cells). It has been difficult in the past to harness the power of mouse genetics to analyze such questions because there was no efficient strategy to disrupt genes selectively in mature T cells (i.e. after thymic egress). Using a novel genetic approach to overcome this limitation, we inactivated the genes encoding Thpok and LRF in post-thymic T cells, and found that the combined activity of both factors is required to maintain the 'integrity' and function of CD4 T cells. Disruption of these factors alters the functional responses of CD4 T cells, as assessed by their cytokine production or expression of surface molecules needed for helper functions. In particular, it impairs the ability of CD4 T cells to produce Type 2 cytokines, including IL-4, which are needed for defenses against helminths, and to mount appropriate responses to an antigenic stimulus. We further found that the impact of Thpok in mature T cells is mediated both by its repression of Runx3 expression and its functional antagonism of Runx protein activity. These findings demonstrate that Thpok and LRF are continuously needed to maintain the integrity of helper T cell responses, in addition to their role in the differentiation of CD4 T cells in the thymus. There results also emphasize the inherent instability of the CD4 lineage, that Thpok and LRF protect from CD8 trans-differentiation. We are currently investigating the mechanistic bases of this critical functions of Thpok and LRF. In addition, the broad impact of Thpok and LRF on T helper functions makes these factors potential targets for physiological control and therapeutic intervention. Notably, CD4 molecules serve in primate T cells as receptors for the human immunodeficiency virus (HIV) and its simian homologs (SIV). Previous studies by our collaborator Jason Benchley (NIAID) had shown that antigen-experienced MHC II-restricted cells convert from a CD4 to a CD8-like phenotype in specific monkey species. Although such conversion is associated with reduced cell intrinsic expression of helper genes, it is believed to be beneficial on a population basis by protecting helper cells from SIV infection and the ensuing immunodeficiency. Our findings provides a conceptual framework to study this conversion, in which the association of functional alterations with changes in coreceptor expression suggests the possible involvement of Runx and Thpok-family transcription factors.